Insoluble aggregates of normally well-behaved proteins are associated with a variety of disease states, including the various forms of amyloidosis, such as Alzheimer's disease, and the prion diseases such as scrapie and bovine spongiform encephalomyelitis, also known as Mad Cow Disease. A family of proteins, called molecular chaperones, exists in cells to overcome the intrinsic propensity of polypeptides to aggregate during the normal folding process. However, under certain circumstances proteins such as the amyloid β (Aβ) peptide associated with Alzheimer's disease form insoluble protein aggregates in spite of the presence of chaperones. In some amyloid diseases, aggregates appear to be toxic simply by virtue of the effect of their accumulated mass in interfering with normal tissue function. In neurodegenerative diseases like Alzheimer's and Huntington's Diseases, the toxic effect appears to be much more subtle. According to one hypothesis, aggregates become toxic when they clog the cell's normal machinery for clearing aggregates and other superfluous proteins. Another hypothesis holds that aggregate toxicity derives from the ability of aggregates to recruit other essential proteins and in the process deplete the normal environment of their activities. In any case, it would be of considerable value to develop ways of removing these aggregates in a benign way, or preventing their formation, in analogy to the actions of molecular chaperones.
Analogously to the situation in Alzheimer's Disease and Aβ peptide, there are at least eight inherited neurodegenerative diseases, including Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy (DRPLA), and spinocerebellar ataxias 1, 2, 3, and 6, that are linked to a particular type of protein aggregate.
Although each of these diseases is associated with a different protein, the proteins share the common feature of containing what is referred to as an expanded polyglutamine (poly(Gln)) repeat. These poly(Gln) expansion-related diseases, often referred to as CAG repeat diseases because the glutamine in the poly(Gln) peptide is coded for by the nucleotides CAG, are progressive disorders characterized by motor and/or cognitive impairments and distinctive pathological patterns of neuronal degeneration. The only mutation implicated in these diseases is an expansion of a poly(Gln) sequence in the disease-related protein, generally from a benign length of less than 37 Gln (also referred to as Q37) to a pathological length of Q37 or more.
All of these neurodegenerative disorders present a common feature: the aggregation of the poly(Gln) repeat disease-related protein into insoluble neuronal intranuclear inclusions, which has become the neuropathological signature of poly(Gln) disorders. The important role that long poly(Gln) repeats play in poly(Gln)-related disorders has been confirmed in a number of models in which mutant forms of various disease proteins were expressed in transgenic mice, Drosophila, or the nematode Caenorhabditis elegans. 
Although these diseases exhibit similar physiological abnormalities, the only common features of disease-related proteins are the poly(Gln) domains. Because of this, the expanded poly(Gln) is believed to be responsible for the pathogenesis. As discussed above, their toxicity is believed to be due to their ability to recruit other critical cellular proteins, via their own poly(Gln) components, into the growing aggregate. The loss of protein activity due to this sequestration appears to be toxic to the cell.
Much about these poly(Gln) diseases remains to be learned. One problem in studying these diseases is that poly(Gln) peptides having a Gln repeat of more than Q35 are poorly soluble when transferred directly into denaturing solvents or water. In some circumstances, such as described in Sharma, FEBS Letters, 456:181–185 (1999), the insolubility of long poly(Gln) repeats has presented such an insurmountable problem that studies had to performed on shorter soluble poly(Gln) repeats such as Q22, even though mutant proteins involved in the spino-cerebellar ataxia type 1 (SCA) are at least Q40.
One method to increase the solubility of monomeric (non-aggregated) Aβ peptide, as disclosed in Evans et al., Proc. Natl. Acad. Sci., 92:763–767 (1995), is to dissolve the peptide in a non-volatile disaggregating solvent such as dimethyl sulfoxide (DMSO). This method has the disadvantage in that the DMSO remains as a permanent co-solvent in the final reaction mixture. Therefore, any results obtained in studies of Aβ peptide dissolved in this way may be biased by the presence of the DMSO.
Use of a volatile disaggregating solvent, trifluoroacetic acid (TFA), to solubilize the Aβ peptide is disclosed in Jao et al., Amyloid: Int. J. Exp. Clin. Invest., 4:240–252 (1997). Volatile disaggregating solvents have an advantage over non-volatile solvents such as DMSO because they are readily removed from the peptide, and thus do not interfere with studies on the peptide. According to this method, TFA is added to the peptide in a glass container at about a 1:1 ratio (ml TFA:mg peptide). Then the TFA and peptide are sonicated, while adding additional TFA, until the peptide completely dissolves. The TFA is then removed with dry nitrogen gas, leaving a coating of peptide on the walls of the container. Trace amounts of the TFA are removed by adding distilled hexafluoroisopropanol (HFIP), sonicating, and removing the HFIP with dry nitrogen gas. Sequential TFA-HFIP treatment has also been disclosed in Zagorski et al., Meths. Enzymol., 309:189–204 (1999). In this protocol, the role played by HFIP seems to be to simply aid in the removal of TFA, which otherwise will make an aqueous solution of the processed peptide acidic and possibly encourage its reaggregation. In our laboratory, this protocol was determined to be effective at solubilizing and disaggregating peptides in the range of Q15–Q35 However, it is poorly effective with peptides greater than Q35.
The inability to dissolve and disaggregate poly(Gln) of the pathological length of Q37 or more represents a major obstacle in studying poly(Gln) diseases. A substantial need exists for a method to solubilize and to disaggregate these peptides, and to maintain these peptides in the disaggregated monomeric state.
Another impediment to the study of poly(Gln) aggregation diseases has been the difficulty in making the aggregates in vitro. Typically, as disclosed by Scherzinger et al., Cell, 90:549–558 (1997) and Scherzinger et al., Proc. Natl. Acad. Sci., 96:4604–4609 (1999), such aggregates are made by recombinantly producing a fusion protein (GST-HDex1) containing glutathione S-transferase and exon 1 of the HD (Huntington's Disease). When the fusion protein is cleaved with trypsin, a high molecular weight protein aggregate with a fibrillar or ribbon-like morphology similar to those found in scrapie and in Alzheimer's disease are formed. These recombinantly produced aggregates do not completely correlate with the natural poly(Gln) disease state.
In the disease state, peptides with poly(Gln) repeat lengths as low as Q15 or Q20, while of insufficient length to readily spontaneously aggregate, readily add to pre-existing aggregates. As disclosed in Perutz, et al., Proc. Natl. Acad. Sci. USA, 91:5355–5358 (1994), aggregates having such shorter poly(Gln) peptides differ from those seen in Alzheimer's disease. Polyglutamine aggregates made in vitro can adopt a number of morphological forms, each of which may differ in toxic activity. Very little is known for certain about the morphology of the toxic form of polyglutamine aggregates in vivo, but if the recruitment hypothesis is correct, than the aggregates must be especially potent in this activity. Consequently, a substantial need exists for methods to prepare different kinds of poly(Gln) aggregates in vitro in order to identify those that are particularly active in supporting deposition and/or extension of additional polyglutamine peptides.
Given the potential role of poly(Gln) aggregates and poly(Gln) aggregate extension in the pathogenesis of expanded CAG repeat diseases, it is essential to characterize the fundamental aggregation behavior of poly(Gln) sequences. Studies of the aggregation behavior dependence on poly(Gln) repeat length are important to fully understand the correlation between length and disease risk, as well as the rules that control the recruitment of other poly(Gln)-containing peptides and proteins into growing poly(Gln) aggregates. Presently, no assay exists that allows the observation of both the homologous growth of an aggregate as well as the ability of the aggregate to recruit other polyglutamine peptides into the aggregate via its extension. Such an assay would have use as an assay for poly(Gln) aggregation and recruitment and as a screen for aggregation inhibitors as potential therapeutics. The assay would also be capable of detecting “extension-competent” or “seeding-competent” aggregates in tissue and serum samples that might be crucial for diagnosis and for evaluating the role of poly(Gln) aggregates in the disease mechanism.